Summary In Xenopus, as in all amphibians and possibly in vertebrate embryos in general, mesoderm formation and the establishment of the dorsoventral axis depend on inductive cell interactions. Molecules involved in mesoderm induction include FGF which acts predominantly as a ventrolateral inducer, the TGF-P homolog activin which can induce all types of mesoderm, and members of the Wnt family which have powerful dorsalizing effects. Early effects of inducer action include the activation of regulatory genes. Among such genes, particular interest is focused on three genes encoding putative transcription factors that are expressed specifically in the Spemann organizer region of the gastrula. Expression of one of these genes, goosecoid, has been shown to be sufficient to elicit the Formation of a dorsal axis including head and notochord in the embryo.
Introduction How does a single cell zygote form an animal with its inultiple tissucs and its morphological organization? This central question in developmental biology has been a major focus of recent research. Among vertebrate systems in which these issues can be addressed, Xenopus laevis is an important example, offering advantages of accessible embryos that can be effectively manipulated, rapid embryogenesis, and a wealth of background information. While details of early development vary between vertebrate orders, the essential features of cleavage and gastrulation are similar. Together with the dramatically growing realization that regulatory molecules and pathways are highly conservative, this similarity provides a basis for applying insights derived in Xenopus to the understanding of embryogenesis in other vertebrates. While embryogenesis in all metazoans shares important features, vertebrate embryos are characterized by a rather high degree of regulative development. By this term we mean the ability of cells and cell groups to change their developmental fate in response to their location and to influences emanating from their neighbors. This article deals with mechanisms of cell interactions in the early Xenopus embryo and the closely linked problem of the determination of the embryonic axes.
The Initial Establishment of Embryonic Axes In its simplest aspect, the embryonic body plan is defined by two axes, the dorsoventral axis and the anteroposterior axis. In XenopiiJ, the oocyte displays one axis. the animal-vegetal axis; this asymmetry is established during oogenesis(1.2).The animal-vegetal axis will be transformed through the complex movements of gastrulation into the anteroposterior axis of the embryo. The unfertilized egg is rotationally symmetrical around its animal-vegetal axis. This symmetry is broken by rotation of the cytoplasm relative to the cortex that is triggered by fertilization (Fig. 1A). This rotation, whose vector is usually set by the site of sperm entry but can be experimentally set at any meridian, determines the position and polarity of the future dorsoventral axis (reviewed in ref. 3). The molccular mechanism of this axis determining event is not understood; a simple hypothetical scenario invokes the movement of dorsalizing substances to the dorsal side of the embryo. While this hypothesis may be too simple to be correct it has been shown that dorsal blastomeres (as the largc cleavage stage cells arc called) are already specified to form dorsal structures and have acquired the ability to induce dorsal structures in neighboring cells. This conclusion is based on experiments where dorsal cells at the 32-cell (Fig. IB) to 64-cell stage were transplanted to ectopic sites. Dorsovegetal blastomeres (e.g., cell D1; sec Fig. 2A) placed at the ventrovegetal position (position D4) can induce a secondary dorsal axis, in effect a second embryo with a head, notochord. somites etc.(hj,while themselves giving rise to endoderm as the fate map predicts (Fig. 2B). More recent studies have shown that all dorsal cells have some capacity of inducing dorsal structures when transplanted to the ventral ~ i d e ' ~ -In~ addition, ). the progeny of dorsal-animal blastomeres (A1, B 1)directly contributes to dorsal structures, even when placed at cctopic sites@). Mesoderm Inducing Factors Whatever the dorsalizing determinant(s) proves to be, it is possible to mimic its effects in several instructive ways. U'hile dorsoventral polarity is established initially in the zygote before first cleavage, it becomes overtly expressed in the polarization of the mesoderm at the beginning of gastrulation (Fig. ID-F). Thus the formation of the mesoderm and the establishment of its polarity are the key to undcrstanding dorsoventral axis formation in the entirc embryo. Mesoderm formation in Xenopus, and probably in all vertebrates, requires cell interactions during cleavage and blastula stages (reviewed in refs. 10-13). As indicated in Fig. lC, signals from the vegetal region induce mesoderm determination in the equatorial region. In the course of this inductive interaction, the mesoderm is polarized along the dorsoventral axis. Many experiments have shown that the endodermal signal is the primary determinant of this polarity, and have led to the hypothesis that at least two (or three) inductive signals arc required, a general mesodennalizing signal and a dorsalizing signal(13,1s).The left drawing in Fig. 1C suggests this dualsignal model. The right drawing in Fig. 1 C suggests an alternative possibility: if the responding cells were different in
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Fig. I . I)cvclopnicnt in Xtvzopiu luwis. Cortical rotation after fertilization (A) blishes the future dorsovcntral polarity. I3y the 32 ccII stage (H), ii dorsalizing center that has inducing ability has formed. Mcsodcrm induction and dorsovcntral axis determination proc(:cd during blastula (C). Polarity rcsults primarily from dorsalizing induction, but is aided by the differential ability to react (competence) ol'dorsal as compared to ventral responding cells. Overt dorsal polarity is expressed with the beginning of gastrulation and the formation 01' tlic dorsal hlnstoporc lip (D). The lip and the advancing dorsal mcsoderm (D-I;), also named the Spcmann organizer, are the source ol' neural inducing signals. With ;idvancement o f Lhc dorsal mesoderm completed (F), the anteroposterior axis ol' the embryo has hccn cstahlishccl. Iilonguf ion accompanied by tissue dil'l'crentiation leads to the swimming tadpole (G). See text for further description.
thcir ability to react, even a homogeneous signal could result i n Ihc induction o f polar mesoderm. A major experimental loo1 for the study of mesoderm induction is illustrated in Fig. 3. Based on earlier work of Nicuwkoop (ref. 10 and citations therein), we know that animal cxplants from blastula embryos differentiate as epidcrinis in control culture (balanced salt solution), but can rcspond (a property called competence in the embryological literature) to mcsoderm inducing signals. Using this assay, rnenibcrs of' the libroblast growth factor (FGF) and transI'orining growth factor beta (TGF-p) families were identified ;IS mcsodcrin induccrs. Several isoforms of FGF arc cff'ective
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as inducers of mostly ventral mesoderm( whilc thc TGF-P family member activin is a potent inducer ol'all ~ y p c s of mcsoderm('X-20,1 1 - 1 3 ) .Activin induces dil'l'crcnt typcs 01' inesodcrm in a concentration-dependent manner("), implying that an activin gradient in the embryo might explain dorsal-to-ventral patterning. This is a very interesting result, but not likely to constitute the entire explanation o f tncso derm induction and polarization, for at least two rcasons, thc short range of inducing signals, and apparent compctciicc differences in responding cells. While the inducing signal exerts primary control in determining the dorsoventral polarity, responding cells arc not equivalent (Fig. 1 C). The extent of dorsal and antcrior differentiation that is achieved by an explant exposcd to activin depends on its size and tlic location within the animal heinisphere from which it is dcrived(22,23)). The simplest version ol' a gradient-dependent polarization assumes a homogeneous population of responding cells, but gradients could also be important in affccting cells that vary in their responsiveness.
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Pig. 2. The 32 cell embryo. A, cell identifications as used in this paper. B, a simplilied Iitc map, bascd on refs. 4,s.
RNA Injection as an Assay for Dorsal Axis Determination The animal explant assay cliscusscd above has hccn very uscful, but has various limitations. An alternative approach is the injection of synthetic mKNA into the fertilized egg or
Mesodermal
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Fig. 3. The mesoderm induction assay. Animal explants follow their inherent fate (Fig. 2) to epidermis in control culture. When
Elongation Tissue Differentiation Gene Activation
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into a blastomere of the early embryo. RNA is usually stable for many hours in this system, and its translation product can act within the context of the whole embryo. If a factor can induce dorsal mesoderm, its ectopic expression on the ventral side of an embryo might elicit the formation of a secondary dorsal axis (Fig. 4). Since activin is a strong inducer of dorsal mesoderm, it is not unexpected that injection of its mRNA can elicit the formation of a secondary axis(20).This axis is, however, quite incomplete, i.e., no fully organized head, or trunk structures with notochord and somites are formed; this result suggests that activin induction alone is not sufficient to set in motion all events necessary for pattern formation. Injection of the mRNA of FGF fails to generate ectopic differentiation of dorsal tissues, as might be expected from its major ventrolateral inducing effects. However, interesting effects of FGF mRNA injection are seen when culturing explants from injected embryos(24). Highly effective axis-inducing molecules belonging to the Wnt family of proteins have been discovered by the RNA injection methodology. The int-1 protooncogene, renamed Wnt-I because of its homology to the Drosophila gene wingless(*j), is the prototype of a family of genes encoding secreted, signal transmitting proteins. Wnt proteins apparently adhere to the cell surface and no soluble preparation is available; yet Wnt can act in a paracrine manner by close cell apposition(26).Thus, the animal explant assay is not applicable to the study of Wnt proteins in mesoderm induction or axis formation. Injection of either mouse Wnt- 1 or Xenopus
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Fig. 4.Jnjection of mRNA as an assay for the dorsalizing effect of various molecules. See text for further discussion.
recombined with vegetal tissue (left), tnesoderm is induced in such explants(lO).Exposure of explants to growth factor? like FGF or activin (right) is an assay for the mesoderm-inducing ability of these factors.
Xwnt-8 mRNA leads to the induction of a complete axis, i.e.. a ‘Siamese twin’ double e m b r y ~ ( ~ ~ .Since ’ ~ ) . Wnt proteins remain closely associated with the producing cells, it is clear that any signal mediated by these proteins can be only short range unless it is relayed. Induction signals may well be short range in general: while FGF and activin can be obtained as soluble molecules, growth factors tend to interact with the extracellular matrix (e.g., ref. 29) and may not freely diffuse in tissues. The dramatic axis-inducing effect of Writ mRNA suggests that one or more members of the Wnt family of genes are involved in mesoderm induction and axis determination. Yet the situation is far from clear. Xwnt-8 is normally expressed at the ventral side of the frog embryo, and its expression is enhanced by ventralizing and repressed by dorsalizing inlervent ion^(^^,^^). Thus, it is unlikely that Xwnt-8 is a major dorsalizing signal in the embryo. It is possible that injected Xwnt-8 mimics the function of another Wnt family protein that might be a natural dorsalizing agent; additional Writ genes are expressed in Xrnopus embryogenesis(7’).Furthermore, additional dorsal axis-inducing molecules unrelated lo the gene families discussed, may have a role in Xenopus embryogenesis. as suggested by Smith and Harland(28).
Mesoderm Induction, the Dorsalizing Center, and the Spemann Organizer Dorsoventral polarity is overtly expressed at the gastrula stage when cell invagination at the dorsal blastopore lip is initiated (Fig. 1D). The lip and advancing dorsal mesoderm (Fig. ID-F) are named the organizcr since Spemann and M a n g ~ l d cshowed ~ ~ ) that transplantation of this region into the ventral side of a host embryo produced a secondary axis. The Spemann organizer has remarkable abilities to self differentiate. induce, organiLe and regulate; it has thus been a focus of interest in embryology for much of this century. As discussed above, secondary axis formation was also achieved by transplantation of cleavage stage blastomercs(@); this region of the early embryo has been called a dorsalizing or Nieuwkoop center(3).The dorsalizing center and Spemann organizer have similar properties, but refer lo different stages of development. A simple, and probably oversimplified, view of the process is that the dorsahing center releases inducing factors (e.g., Wnt, activin, and probably others) inducing the dorsal-equatorial region of the embryo to become organizer tissue. At the beginning of gastiulation, cell movements lead to
Fig. 5. Whole mount in situ hybridizati~ni~~’ of gastrula embryo with Xlim-1 probe. Localization of the RNA, visualized by dark staining, i s seen in the dorsal blastopore lip and dorsal mesoderm. The stage and orientation of this embryo is similar to the drawing i n Fig. 1E. Figure from ref. 37.
the elongation and migration of dorsal mesoderm along the roof of the blastocoel (Fig. 1D-F; ref. 33), establishing the axial mesoderm (notochord and somites). In addition, inductive influences emanating from the region of the dorsal lip and the advancing mesoderm reprogram the overlying ectoderm to become the neural plate; the anteroposterior axis of the nervouh \ystcm and of the entire animal is established in the course of this process (reviewed in ref. 34). The axis forming potency of the organiLer region is an expression, under experimental conditions, of the patterning function of this tissue in normal embryogenesis. In other words, transplantation experiments and observation of normal gastrulation indicate that the organizer region is primarily responsible for controlling the formation of the nervous system and the establishment of thc anteroposterior polarity of the embryo. For these reasons, the isolation of molecular markers for the organizer as a basis for studying the mechanism of its developmcntal function is a subject of great interest.
Responses to Induction: Expression of Organizer Specific Regulatory Genes Three genes encoding putative transcription factors have been isolated that are expressed predominantly or exclusively in the organizer region of Xenopus gastrulae. They include the homeobox genes goosecoid(3s,36)and Xlim-l(37), and XFKH-li38’, showing homology to the forkhead domain of regulatory genes in Drosophila and mammals. The localization of Xlim-1 RNA in the dorsal mesoderm of a midgastmla embryo is illustrated in Fig. 5. Transcripts of these genes are present at a low level if at all in maternal RNA, and begin accumulation at or shortly after the midblastula stage
(the socalled midblastula transition, when embryonic transcription is initiated). RNA concentrations peak at gastrula and then decrease; expression patterns at later phases have not yet been fully explored, but certainly vary considcrably between these three gcnes. All three genes are induced by activin in animal explants in a manner that appears to be independent of protein synthesis, signifying that the activation of these genes is an early response to mesoderm induction. An important difference in the behavior of goosecoid and Xlim-1 is seen in their response to retinoic acid (RA) and I,i+. RA has many interesting effects in development. When applied to whole Xenopus embryos it leads to inhibition of anterior development, at high levels to headless tadpoles(39). In explant experiments, K A suppresses the dorsal and enhances the ventral character of the tissue(40);because of the role of dorsal mesoderm as inducer of the anterior nervous system (see Fig. l),inhibition of dorsal and anterior devclopment may be two aspects of the same action of RA. Li+ can dorsalize embryos, leading to exaggerated development of dorsoanterior structures (e.g., cement gland, forebrain. eyes) by transforming the entire mesoderm into dorsal mesoderm, i.e., organizer(“). The response of goosecoid is consistent with its nature as a marker for the organizer: its cxpression is enhanced by Li+treatment, while RA suppresses the activinmediated induction of this gene(36).In contrast, Xlinz-I docs not respond to Li+ treatment and is induced in animal explants by KA; activin and RA have a syncrgistic effect(37). There is no simple explanation available for these results, but they make it unlikely that a gradient of RA is a direct cause of dorsoventral polarity in the embryo. The goosecoid gene is not only a marker for the organizer, but can elicit the formation of the organizer. By injecting synthetic goosecoid mRNA into ventral blastomeres (see Fig. 4), Cho et al.c3@ achieved the formation of sccondary axes (with a head, notochord, etc.). This result suggests that the goosecoid product is either at the top or within a unique path of the regulatory hierarchy required for axis formation, so that its ectopic expression can set in motion all subsidiary steps sufficient for the production of an axis. These experiments complement the results of injection of mRNA encoding inducing factors, primarily Wnt, as discussed earlier. It appears likely that the ectopic expression of an inducer like Wnt elicits the expression of the goosecoid gene, and likely other genes, in adjacent cells; direct ectopic expression of goosccoid bypasses the requirement for the initial inducing signal and leads more directly into the dorsal axis-forming pathway. Genes expressed in the organizer region are certainly not the only regulatory genes activated in mesoderm induction. Other genes of particular interest include ‘pan-mesodermal’ genes like the early response gene Mix. 1, whose expression domain also includes the future endoderm and whose time of expression is limited to a short period during gastrulation(43), and Xbra, the homolog of the mouse brachyury gene(44).In addition, cell type-specific genes are activated as a subsequent response to mesoderm induction, notably myoD which precisely delineates the muscle-forming region of the embryo(45!. Several other genes of interest have been
described, yet it is undoubtedly true that many of the key players during early Xenopus embryogenesis remain to be discovered.
Conclusions Mesoderm formation and axis establishment in the Xenopus ernbyo begin with maternal information laid down in the egg; proceed through inductive influcnces during the cleavage and blastula stages that change the determination state of equatorial region cells; and become ovcrt with thc beginning of gastrulation. Several peptide growth factors have been identified that can transmit signals in the embryo. Cells responding to inductive signals do several things: they change their ability to respond (competence) to additional signals; they send out signals of their own; they change their pattern of gene expression; and, not discussed here but undoubtedly important, they.change their adhesive propertics. The transcriptional rcsponse has been studied exlcnsively, and several regulatory genes activated by induction have been identified. Among these, particular interest is focused on genes expressed in the organizer region, the region responsible for establishing the dorsal axis of the embryo and, ultimatcly, thc body pattern of the organism. References 1Hausen,P.snd Riebesell,M. (1991).The EurlvDeveZopnientofXenopusheits. 142 pp. Springcr Vcrlag. Berlin. 2 Danilehik, M. V. and Cerhart, J. C. (1987).Differentiatinn of the animal-vegetal axis i n Xenopus fuevis oocytes: 1. Polarized intracellular translocation of platelets estiblishes the yolkgradient. Drv. Biol. 122,101-I12. 3 Gerhart, J.. Danilehik, M., Doniach, T., Roberts, S., Rowning, B. and Stewart, R. (1989).Cortical rotation or the Xenopus egg: cunsequences for the anteroposterior pattern of embryonic dorsal development. Developrnrnt 107Supplement. 37-51. Fate map ofthc 32-ccllstage of Xenopirs luevis. 4 Dale, L.and Slack, J. Y.W. (.1987). Develapmrru 99.527-55I . 5 Moody, S. A. (1987).Fates of the blastomeres of the 32-cell-stageXennpus embryo. Dev. B i d . 122.300-319. 6 Cimlich, R. L. and Gerhart, J. C. (1984). Early cellular interactions prumote cmbryonic axis formation in Xenopus Iuevis. Dev. Biol. 104, 117-130. 7 Gimlich, R. L. (1986).Acquisition of developmcntal autonomy in the cquatorial region of the Xenopus cmbryo. Dev. B i d . 115,340-352. 8 Kageura, H. (1990). Spatial dislribulion of the capacity to initiate a secondary emhryo in the 32-cellembryo of Xeiiopus laevis. Dev. Biol. 142,432-338. 9 Gallagher, B. C., Hainski, A. Y. and Moody, S. A. (1991).Autonomous ditferentiationof dorsal axial structurcs from an animal cap cleavage blaslomeres in Xenopus. Deirlopinenr 112,I 103-11 14. 10 Nienwkoop, P.D. (1973).The "organisation center" of the amphibian embryo: its origin,hpatial organization. and morphogenetic action. Adv. Mnrphogener. 10.1-39. 11 Smith, J. C. (1989).Mesoderm induction and mcsodcrm-inducing factors in early amphibian development. Developnreiit 105.665-677. 12 Dawid, I. B., Sargent, T. D. and Rosa, F. (1 990). The role of growth factors in cmbryonic induction in aniphihianh. C.'urr. Topics in Deuel. B i d . 24,261-288. T.M.and Melton, I).k (1992).Diffusible factors in vcrtcbratc cmbryonic 13J-I, induction.Cell 68.257-170. 14 Dale, L. and Slack, J. M. W. ( 1987).Regionid specification within the mesoderm orearly embryos in Xenopus hevis. Development 100,279-295. 15 Smith, J. C. and Slaek, J. M. W. (19831.Dorsalization and neural induction: propertiesof the organizerin Xrrwprs luevis. .I. E n i h y l . Exp. Morphol. 78,299-317. 16 Slack, J. M. W, Darlington, li. G., Heath. J. K.and Codsave, S. F. (1987). Mesoderm induction in early Xenopus embryos by hcparin-binding growth factors. Nature 326.197-200. 17 Kimelman,D. and Kirsebner, M. (1987).Synergistic induction of mesoderm by FGF and TGF-bela and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51,869-877. 111 A s a s h i , M., Nakano, It,Shlmada, K., Kinoshita, K.,Isbii, K.,Shibai, H. and Ueno, N. (1990).Mesdemal induction in early amphibian embryos by activin A (erythrniddifferentiationfktor). Roux's Arch. Dm. Biol.198.330-335. 19 Smith, J. C.,price, 8. M. J., Van Nimmen, g.and Huylebmck, D. (1990). Idcntification of a pnienl Xenopzrs mesoderm-inducing factor as a homologuc of activin A. Nirrure 345.7119-731.
20 Thomwn, G., WnoK, T.,Whitman. M.,Sokol, S., Vaughan, J., Vale, W. and Melton. D. A. ( 1990).Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm andantcrior structures. Cell 63.485493. 21 Green, .I. R. and Smith. J. C.('1990). Gradcd changes in dose of a Xenopus aclivin homologue elicit stepwise transitions in embryonic cell fate. Nurure 347,391-394. 22 Sokol, S. and Melton. D. A. (1991J. Preexisting pattern in Xenopus animal polc cdls revealed by induction with activin. Nnture 351,409-411. 23 Dawid, 1. R.. Taira, M.,Good, P.J. and Rebagliati, M. R. (1992).The role of growth factorsin embryonic induction in Xen0pu.s lueris. Mol. Reprod. Dev.. in press. 24 Kmelman, D. and Mars. A. (1992). Induction of dorsal and venlral mesoderm hy ectopically expressed Xenopus basic fibroblast growth factor. k e l o p n i e n t 114,261260. 25 Nusse, R.,Brown, A..Papkoff, J., Scambler, P..Shackleford, G.,McMahon. A., Moon,R.andVarmu..,H.(1991).Ancwnomenclatureforinf-l andrelatedgenes: the Wnfgene family. Ce16 64,231 . 26 Jue, S. F., Bradlcy. R S., Rudnicki. J. A.. Varmus, H. E. and Brawn, A. M.C. (1992).The mouse Wnl-1 gene can act via a paracrinc mcchanism in transformation of mammary epithelial cells. Mol. Cel6. Eiol. 12,321-328. 27 Sokol, S., Christian, J. L., Moon, R. T.and Melton. D. A. (1991).lnjected Wnt RNA inducesa complete body axis in Xenopus emhryoa. Cell 76,741-75?, 28 Smith, W. C. and Harland. R. M. (1991). Injected Xwnt-8 KNA Xenopus emhryos to promote formation of a vcgctal dorsalizing center. Cell 67.753765. 29 Baird, A. and Bohlen. P. (1990).Fibroblast growth factors. In Peptide tirowth Fucmrs and fiirir Rrceptor.s I (eds. M. B. Sporn and A. B. Roberts). pp 369418. Springer Verlag. Berlin. 30 Christian, J. L., McMahon, J. A., MrMahon, A. P.and Muun, R. T. ( 1991). XM'llf-8.a Xenopus Wnf-l/inf-l related genc rcsponsivc to mesoderm inducing factors, IMY play a role in ventral mesodermal patterning during embryogenesis. Drvdoprnenf 111.1045-1055. 31 Christian,J. L.. Cavin, B.J., McMahon, A. P.and Moon, R. T.(1991 I. Isolation of cDNAs partially cncoding four wnt-Mnt-1 rclatcd proteins and characterization of their transient expression during embryonic develnpmenl. /lev.B i d . 143,230-234. 32 Spemann, H. und Mangold. H. (1924). Uber lnduktion von Bmbryonalanldgen durch Implantation artfremder Organisatoren. Wiulelrn Roux' Arch. Enrnicklungsmech. Org. 100,599-638. 33 Keller. R (1991).h y embryonic development of Xenopus lamis. MefhodsCell BioZ.. vol. 36,(eds. B. K.Kay and H. B. Peng), pp 61-113. Academic Press, San Diego. 34 Sharpe, C. R (1990).kgional neural induction in Xrrmpus laevis. BioE.ssays 12. s91-596. 35 Blumberg, B.. Wright, C. V. E.. De Robertis, E. M. and Cho, K.W. Y.(1991J. Organizcr-specific homeohx genes i n X e i u p s luevis embryos. Scirnce 253. 194-196. 36 Cho. K.W.Y.,Blumherg, B., Steinbeisser, H. and De R o b e m , E. M. (1991). Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goo,Oremid.Cell 67,1 11 1 - 1 1 20. 37 Taus, M..Jamrich, M., Good. P. J. and Dawid. 1. B. (1992).The LIM domaincontaining homeobox gcnc XZinr-J is cxprcsscd spccitically in thc organizer region of Xenopur gastrula emhryos. Genes 1)C.i~.6.356-366. 38 Dirksen, M.L.and Jamrich,M. (1992).A novel, activin inducible, blastopore lip spccific gcnc ofXenopus Zaevis contains a,forkheudDNAbinding domain. Genes. Ilev. 6.599-608. 39 Dunton, A. J., ' h e r m a n s . J. P.M., Hage, W. J., Hendriks,H. F. J., de Vries, N. J.. Heideveld. M. and Nieuwkoop, P. D. (19891.Rrtinoic acid causes mi anteroposterior trans~omr;llionin lire developing cenlral nervous system. Nozure 340. 140-144. 40 Kuiz i Altaha, A. and Jessell, T. (1991).Rctinoic acid modifics mesodermal pattcrning in carly Xenopus embryos. Genes Ilcv. 5. 175-187. 41 Kao, K.R. and Elinson, R. P. (1988).The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus lurvis embryos. Der: Biol. 127,64-77. 42 Hemmati-Brivanlou, A.. Frank, D., Bolee. ME., Brown, B.D., Sive, H.I.., and Harland, R.M. (1990). I.ncali/alion of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. DewZopnzent 110.325-330. 43 Rosa, F.M. (1989).Mix.1. an homcobox mRNA inducible by m e s h 1 i n d u m , is expressed mostly in the presumptive endodermal cells orxenopus embryos. Cell 57, 965-974. 44 Smith, J. C., Price, B. M.J, Green, J. B. A., Weigel, D. and Herrmann. R.G. (1991).Expression of a Xenopus homolog or Brnchyuv (T)is an immediate early response to mesoderm induction. Cell 67,7947. 45 Hopwood, N. D.,Huek, A. and Guidon, ,I. R. (1989). MyoU expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. fi.'MBU J. 8.3W-3417.
Igor B.Dawid is at the NICHHD. Building 6B, Rm.41 3, NIH, Bcthesda, MD 20892, USA. I
I
Introduction How does a single cell zygote form an animal with its inultiple tissucs and its morphological organization? This central question in developmental biology has been a major focus of recent research. Among vertebrate systems in which these issues can be addressed, Xenopus laevis is an important example, offering advantages of accessible embryos that can be effectively manipulated, rapid embryogenesis, and a wealth of background information. While details of early development vary between vertebrate orders, the essential features of cleavage and gastrulation are similar. Together with the dramatically growing realization that regulatory molecules and pathways are highly conservative, this similarity provides a basis for applying insights derived in Xenopus to the understanding of embryogenesis in other vertebrates. While embryogenesis in all metazoans shares important features, vertebrate embryos are characterized by a rather high degree of regulative development. By this term we mean the ability of cells and cell groups to change their developmental fate in response to their location and to influences emanating from their neighbors. This article deals with mechanisms of cell interactions in the early Xenopus embryo and the closely linked problem of the determination of the embryonic axes.
The Initial Establishment of Embryonic Axes In its simplest aspect, the embryonic body plan is defined by two axes, the dorsoventral axis and the anteroposterior axis. In XenopiiJ, the oocyte displays one axis. the animal-vegetal axis; this asymmetry is established during oogenesis(1.2).The animal-vegetal axis will be transformed through the complex movements of gastrulation into the anteroposterior axis of the embryo. The unfertilized egg is rotationally symmetrical around its animal-vegetal axis. This symmetry is broken by rotation of the cytoplasm relative to the cortex that is triggered by fertilization (Fig. 1A). This rotation, whose vector is usually set by the site of sperm entry but can be experimentally set at any meridian, determines the position and polarity of the future dorsoventral axis (reviewed in ref. 3). The molccular mechanism of this axis determining event is not understood; a simple hypothetical scenario invokes the movement of dorsalizing substances to the dorsal side of the embryo. While this hypothesis may be too simple to be correct it has been shown that dorsal blastomeres (as the largc cleavage stage cells arc called) are already specified to form dorsal structures and have acquired the ability to induce dorsal structures in neighboring cells. This conclusion is based on experiments where dorsal cells at the 32-cell (Fig. IB) to 64-cell stage were transplanted to ectopic sites. Dorsovegetal blastomeres (e.g., cell D1; sec Fig. 2A) placed at the ventrovegetal position (position D4) can induce a secondary dorsal axis, in effect a second embryo with a head, notochord. somites etc.(hj,while themselves giving rise to endoderm as the fate map predicts (Fig. 2B). More recent studies have shown that all dorsal cells have some capacity of inducing dorsal structures when transplanted to the ventral ~ i d e ' ~ -In~ addition, ). the progeny of dorsal-animal blastomeres (A1, B 1)directly contributes to dorsal structures, even when placed at cctopic sites@). Mesoderm Inducing Factors Whatever the dorsalizing determinant(s) proves to be, it is possible to mimic its effects in several instructive ways. U'hile dorsoventral polarity is established initially in the zygote before first cleavage, it becomes overtly expressed in the polarization of the mesoderm at the beginning of gastrulation (Fig. ID-F). Thus the formation of the mesoderm and the establishment of its polarity are the key to undcrstanding dorsoventral axis formation in the entirc embryo. Mesoderm formation in Xenopus, and probably in all vertebrates, requires cell interactions during cleavage and blastula stages (reviewed in refs. 10-13). As indicated in Fig. lC, signals from the vegetal region induce mesoderm determination in the equatorial region. In the course of this inductive interaction, the mesoderm is polarized along the dorsoventral axis. Many experiments have shown that the endodermal signal is the primary determinant of this polarity, and have led to the hypothesis that at least two (or three) inductive signals arc required, a general mesodennalizing signal and a dorsalizing signal(13,1s).The left drawing in Fig. 1C suggests this dualsignal model. The right drawing in Fig. 1 C suggests an alternative possibility: if the responding cells were different in
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Fig. I . I)cvclopnicnt in Xtvzopiu luwis. Cortical rotation after fertilization (A) blishes the future dorsovcntral polarity. I3y the 32 ccII stage (H), ii dorsalizing center that has inducing ability has formed. Mcsodcrm induction and dorsovcntral axis determination proc(:cd during blastula (C). Polarity rcsults primarily from dorsalizing induction, but is aided by the differential ability to react (competence) ol'dorsal as compared to ventral responding cells. Overt dorsal polarity is expressed with the beginning of gastrulation and the formation 01' tlic dorsal hlnstoporc lip (D). The lip and the advancing dorsal mcsoderm (D-I;), also named the Spcmann organizer, are the source ol' neural inducing signals. With ;idvancement o f Lhc dorsal mesoderm completed (F), the anteroposterior axis ol' the embryo has hccn cstahlishccl. Iilonguf ion accompanied by tissue dil'l'crentiation leads to the swimming tadpole (G). See text for further description.
thcir ability to react, even a homogeneous signal could result i n Ihc induction o f polar mesoderm. A major experimental loo1 for the study of mesoderm induction is illustrated in Fig. 3. Based on earlier work of Nicuwkoop (ref. 10 and citations therein), we know that animal cxplants from blastula embryos differentiate as epidcrinis in control culture (balanced salt solution), but can rcspond (a property called competence in the embryological literature) to mcsoderm inducing signals. Using this assay, rnenibcrs of' the libroblast growth factor (FGF) and transI'orining growth factor beta (TGF-p) families were identified ;IS mcsodcrin induccrs. Several isoforms of FGF arc cff'ective
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as inducers of mostly ventral mesoderm( whilc thc TGF-P family member activin is a potent inducer ol'all ~ y p c s of mcsoderm('X-20,1 1 - 1 3 ) .Activin induces dil'l'crcnt typcs 01' inesodcrm in a concentration-dependent manner("), implying that an activin gradient in the embryo might explain dorsal-to-ventral patterning. This is a very interesting result, but not likely to constitute the entire explanation o f tncso derm induction and polarization, for at least two rcasons, thc short range of inducing signals, and apparent compctciicc differences in responding cells. While the inducing signal exerts primary control in determining the dorsoventral polarity, responding cells arc not equivalent (Fig. 1 C). The extent of dorsal and antcrior differentiation that is achieved by an explant exposcd to activin depends on its size and tlic location within the animal heinisphere from which it is dcrived(22,23)). The simplest version ol' a gradient-dependent polarization assumes a homogeneous population of responding cells, but gradients could also be important in affccting cells that vary in their responsiveness.
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Pig. 2. The 32 cell embryo. A, cell identifications as used in this paper. B, a simplilied Iitc map, bascd on refs. 4,s.
RNA Injection as an Assay for Dorsal Axis Determination The animal explant assay cliscusscd above has hccn very uscful, but has various limitations. An alternative approach is the injection of synthetic mKNA into the fertilized egg or
Mesodermal
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Fig. 3. The mesoderm induction assay. Animal explants follow their inherent fate (Fig. 2) to epidermis in control culture. When
Elongation Tissue Differentiation Gene Activation
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into a blastomere of the early embryo. RNA is usually stable for many hours in this system, and its translation product can act within the context of the whole embryo. If a factor can induce dorsal mesoderm, its ectopic expression on the ventral side of an embryo might elicit the formation of a secondary dorsal axis (Fig. 4). Since activin is a strong inducer of dorsal mesoderm, it is not unexpected that injection of its mRNA can elicit the formation of a secondary axis(20).This axis is, however, quite incomplete, i.e., no fully organized head, or trunk structures with notochord and somites are formed; this result suggests that activin induction alone is not sufficient to set in motion all events necessary for pattern formation. Injection of the mRNA of FGF fails to generate ectopic differentiation of dorsal tissues, as might be expected from its major ventrolateral inducing effects. However, interesting effects of FGF mRNA injection are seen when culturing explants from injected embryos(24). Highly effective axis-inducing molecules belonging to the Wnt family of proteins have been discovered by the RNA injection methodology. The int-1 protooncogene, renamed Wnt-I because of its homology to the Drosophila gene wingless(*j), is the prototype of a family of genes encoding secreted, signal transmitting proteins. Wnt proteins apparently adhere to the cell surface and no soluble preparation is available; yet Wnt can act in a paracrine manner by close cell apposition(26).Thus, the animal explant assay is not applicable to the study of Wnt proteins in mesoderm induction or axis formation. Injection of either mouse Wnt- 1 or Xenopus
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Fig. 4.Jnjection of mRNA as an assay for the dorsalizing effect of various molecules. See text for further discussion.
recombined with vegetal tissue (left), tnesoderm is induced in such explants(lO).Exposure of explants to growth factor? like FGF or activin (right) is an assay for the mesoderm-inducing ability of these factors.
Xwnt-8 mRNA leads to the induction of a complete axis, i.e.. a ‘Siamese twin’ double e m b r y ~ ( ~ ~ .Since ’ ~ ) . Wnt proteins remain closely associated with the producing cells, it is clear that any signal mediated by these proteins can be only short range unless it is relayed. Induction signals may well be short range in general: while FGF and activin can be obtained as soluble molecules, growth factors tend to interact with the extracellular matrix (e.g., ref. 29) and may not freely diffuse in tissues. The dramatic axis-inducing effect of Writ mRNA suggests that one or more members of the Wnt family of genes are involved in mesoderm induction and axis determination. Yet the situation is far from clear. Xwnt-8 is normally expressed at the ventral side of the frog embryo, and its expression is enhanced by ventralizing and repressed by dorsalizing inlervent ion^(^^,^^). Thus, it is unlikely that Xwnt-8 is a major dorsalizing signal in the embryo. It is possible that injected Xwnt-8 mimics the function of another Wnt family protein that might be a natural dorsalizing agent; additional Writ genes are expressed in Xrnopus embryogenesis(7’).Furthermore, additional dorsal axis-inducing molecules unrelated lo the gene families discussed, may have a role in Xenopus embryogenesis. as suggested by Smith and Harland(28).
Mesoderm Induction, the Dorsalizing Center, and the Spemann Organizer Dorsoventral polarity is overtly expressed at the gastrula stage when cell invagination at the dorsal blastopore lip is initiated (Fig. 1D). The lip and advancing dorsal mesoderm (Fig. ID-F) are named the organizcr since Spemann and M a n g ~ l d cshowed ~ ~ ) that transplantation of this region into the ventral side of a host embryo produced a secondary axis. The Spemann organizer has remarkable abilities to self differentiate. induce, organiLe and regulate; it has thus been a focus of interest in embryology for much of this century. As discussed above, secondary axis formation was also achieved by transplantation of cleavage stage blastomercs(@); this region of the early embryo has been called a dorsalizing or Nieuwkoop center(3).The dorsalizing center and Spemann organizer have similar properties, but refer lo different stages of development. A simple, and probably oversimplified, view of the process is that the dorsahing center releases inducing factors (e.g., Wnt, activin, and probably others) inducing the dorsal-equatorial region of the embryo to become organizer tissue. At the beginning of gastiulation, cell movements lead to
Fig. 5. Whole mount in situ hybridizati~ni~~’ of gastrula embryo with Xlim-1 probe. Localization of the RNA, visualized by dark staining, i s seen in the dorsal blastopore lip and dorsal mesoderm. The stage and orientation of this embryo is similar to the drawing i n Fig. 1E. Figure from ref. 37.
the elongation and migration of dorsal mesoderm along the roof of the blastocoel (Fig. 1D-F; ref. 33), establishing the axial mesoderm (notochord and somites). In addition, inductive influences emanating from the region of the dorsal lip and the advancing mesoderm reprogram the overlying ectoderm to become the neural plate; the anteroposterior axis of the nervouh \ystcm and of the entire animal is established in the course of this process (reviewed in ref. 34). The axis forming potency of the organiLer region is an expression, under experimental conditions, of the patterning function of this tissue in normal embryogenesis. In other words, transplantation experiments and observation of normal gastrulation indicate that the organizer region is primarily responsible for controlling the formation of the nervous system and the establishment of thc anteroposterior polarity of the embryo. For these reasons, the isolation of molecular markers for the organizer as a basis for studying the mechanism of its developmcntal function is a subject of great interest.
Responses to Induction: Expression of Organizer Specific Regulatory Genes Three genes encoding putative transcription factors have been isolated that are expressed predominantly or exclusively in the organizer region of Xenopus gastrulae. They include the homeobox genes goosecoid(3s,36)and Xlim-l(37), and XFKH-li38’, showing homology to the forkhead domain of regulatory genes in Drosophila and mammals. The localization of Xlim-1 RNA in the dorsal mesoderm of a midgastmla embryo is illustrated in Fig. 5. Transcripts of these genes are present at a low level if at all in maternal RNA, and begin accumulation at or shortly after the midblastula stage
(the socalled midblastula transition, when embryonic transcription is initiated). RNA concentrations peak at gastrula and then decrease; expression patterns at later phases have not yet been fully explored, but certainly vary considcrably between these three gcnes. All three genes are induced by activin in animal explants in a manner that appears to be independent of protein synthesis, signifying that the activation of these genes is an early response to mesoderm induction. An important difference in the behavior of goosecoid and Xlim-1 is seen in their response to retinoic acid (RA) and I,i+. RA has many interesting effects in development. When applied to whole Xenopus embryos it leads to inhibition of anterior development, at high levels to headless tadpoles(39). In explant experiments, K A suppresses the dorsal and enhances the ventral character of the tissue(40);because of the role of dorsal mesoderm as inducer of the anterior nervous system (see Fig. l),inhibition of dorsal and anterior devclopment may be two aspects of the same action of RA. Li+ can dorsalize embryos, leading to exaggerated development of dorsoanterior structures (e.g., cement gland, forebrain. eyes) by transforming the entire mesoderm into dorsal mesoderm, i.e., organizer(“). The response of goosecoid is consistent with its nature as a marker for the organizer: its cxpression is enhanced by Li+treatment, while RA suppresses the activinmediated induction of this gene(36).In contrast, Xlinz-I docs not respond to Li+ treatment and is induced in animal explants by KA; activin and RA have a syncrgistic effect(37). There is no simple explanation available for these results, but they make it unlikely that a gradient of RA is a direct cause of dorsoventral polarity in the embryo. The goosecoid gene is not only a marker for the organizer, but can elicit the formation of the organizer. By injecting synthetic goosecoid mRNA into ventral blastomeres (see Fig. 4), Cho et al.c3@ achieved the formation of sccondary axes (with a head, notochord, etc.). This result suggests that the goosecoid product is either at the top or within a unique path of the regulatory hierarchy required for axis formation, so that its ectopic expression can set in motion all subsidiary steps sufficient for the production of an axis. These experiments complement the results of injection of mRNA encoding inducing factors, primarily Wnt, as discussed earlier. It appears likely that the ectopic expression of an inducer like Wnt elicits the expression of the goosecoid gene, and likely other genes, in adjacent cells; direct ectopic expression of goosccoid bypasses the requirement for the initial inducing signal and leads more directly into the dorsal axis-forming pathway. Genes expressed in the organizer region are certainly not the only regulatory genes activated in mesoderm induction. Other genes of particular interest include ‘pan-mesodermal’ genes like the early response gene Mix. 1, whose expression domain also includes the future endoderm and whose time of expression is limited to a short period during gastrulation(43), and Xbra, the homolog of the mouse brachyury gene(44).In addition, cell type-specific genes are activated as a subsequent response to mesoderm induction, notably myoD which precisely delineates the muscle-forming region of the embryo(45!. Several other genes of interest have been
described, yet it is undoubtedly true that many of the key players during early Xenopus embryogenesis remain to be discovered.
Conclusions Mesoderm formation and axis establishment in the Xenopus ernbyo begin with maternal information laid down in the egg; proceed through inductive influcnces during the cleavage and blastula stages that change the determination state of equatorial region cells; and become ovcrt with thc beginning of gastrulation. Several peptide growth factors have been identified that can transmit signals in the embryo. Cells responding to inductive signals do several things: they change their ability to respond (competence) to additional signals; they send out signals of their own; they change their pattern of gene expression; and, not discussed here but undoubtedly important, they.change their adhesive propertics. The transcriptional rcsponse has been studied exlcnsively, and several regulatory genes activated by induction have been identified. Among these, particular interest is focused on genes expressed in the organizer region, the region responsible for establishing the dorsal axis of the embryo and, ultimatcly, thc body pattern of the organism. References 1Hausen,P.snd Riebesell,M. (1991).The EurlvDeveZopnientofXenopusheits. 142 pp. Springcr Vcrlag. Berlin. 2 Danilehik, M. V. and Cerhart, J. C. (1987).Differentiatinn of the animal-vegetal axis i n Xenopus fuevis oocytes: 1. Polarized intracellular translocation of platelets estiblishes the yolkgradient. Drv. Biol. 122,101-I12. 3 Gerhart, J.. Danilehik, M., Doniach, T., Roberts, S., Rowning, B. and Stewart, R. (1989).Cortical rotation or the Xenopus egg: cunsequences for the anteroposterior pattern of embryonic dorsal development. Developrnrnt 107Supplement. 37-51. Fate map ofthc 32-ccllstage of Xenopirs luevis. 4 Dale, L.and Slack, J. Y.W. (.1987). Develapmrru 99.527-55I . 5 Moody, S. A. (1987).Fates of the blastomeres of the 32-cell-stageXennpus embryo. Dev. B i d . 122.300-319. 6 Cimlich, R. L. and Gerhart, J. C. (1984). Early cellular interactions prumote cmbryonic axis formation in Xenopus Iuevis. Dev. Biol. 104, 117-130. 7 Gimlich, R. L. (1986).Acquisition of developmcntal autonomy in the cquatorial region of the Xenopus cmbryo. Dev. B i d . 115,340-352. 8 Kageura, H. (1990). Spatial dislribulion of the capacity to initiate a secondary emhryo in the 32-cellembryo of Xeiiopus laevis. Dev. Biol. 142,432-338. 9 Gallagher, B. C., Hainski, A. Y. and Moody, S. A. (1991).Autonomous ditferentiationof dorsal axial structurcs from an animal cap cleavage blaslomeres in Xenopus. Deirlopinenr 112,I 103-11 14. 10 Nienwkoop, P.D. (1973).The "organisation center" of the amphibian embryo: its origin,hpatial organization. and morphogenetic action. Adv. Mnrphogener. 10.1-39. 11 Smith, J. C. (1989).Mesoderm induction and mcsodcrm-inducing factors in early amphibian development. Developnreiit 105.665-677. 12 Dawid, I. B., Sargent, T. D. and Rosa, F. (1 990). The role of growth factors in cmbryonic induction in aniphihianh. C.'urr. Topics in Deuel. B i d . 24,261-288. T.M.and Melton, I).k (1992).Diffusible factors in vcrtcbratc cmbryonic 13J-I, induction.Cell 68.257-170. 14 Dale, L. and Slack, J. M. W. ( 1987).Regionid specification within the mesoderm orearly embryos in Xenopus hevis. Development 100,279-295. 15 Smith, J. C. and Slaek, J. M. W. (19831.Dorsalization and neural induction: propertiesof the organizerin Xrrwprs luevis. .I. E n i h y l . Exp. Morphol. 78,299-317. 16 Slack, J. M. W, Darlington, li. G., Heath. J. K.and Codsave, S. F. (1987). Mesoderm induction in early Xenopus embryos by hcparin-binding growth factors. Nature 326.197-200. 17 Kimelman,D. and Kirsebner, M. (1987).Synergistic induction of mesoderm by FGF and TGF-bela and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51,869-877. 111 A s a s h i , M., Nakano, It,Shlmada, K., Kinoshita, K.,Isbii, K.,Shibai, H. and Ueno, N. (1990).Mesdemal induction in early amphibian embryos by activin A (erythrniddifferentiationfktor). Roux's Arch. Dm. Biol.198.330-335. 19 Smith, J. C.,price, 8. M. J., Van Nimmen, g.and Huylebmck, D. (1990). Idcntification of a pnienl Xenopzrs mesoderm-inducing factor as a homologuc of activin A. Nirrure 345.7119-731.
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Igor B.Dawid is at the NICHHD. Building 6B, Rm.41 3, NIH, Bcthesda, MD 20892, USA. I
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